are Gram-negative bacteria that are a normal constituent of the intestinal tract microbiota of humans and warm-blooded animals. Some pathogenic strains of enterotoxigenic E. coli
(ETEC) cause diarrheal disease with severe symptoms and dehydration, leading to annual mortality of hundreds of thousand children below the age of five in developing countries 1
. Our studies investigate the structural features of the archetype ETEC pilus, CFA/I, which promotes host/pathogen interactions, thereby initiating infection. The aim of this research is to reveal structural clues for the development of novel drugs against this critical ETEC virulence factor.
Adhesion pili are specialized to sustain attachment of bacterial cells under the environmental conditions surrounding their preferred host target tissue. For ETEC, this environment is the small intestine. Fluid in the small intestine contains a mixture of solutes, e.g., carbohydrates, peptides and lipids from ingested food, and secretions from the biliary tree and pancreas that contain various digestive enzymes, e.g., electrolytes and pH-regulating substances. The intestinal wall has longitudinal and circular smooth muscle layers that provide mixing and propulsive movement of this chyme for efficient digestion. Contractile rings interspaced along the intestine segment the chyme into compartments and create a circular motion of the fluid. The propulsive movement results in forward fluid movement, and simulations have shown that the contraction phase of the peristaltic reflex generates pressure, shear stress, and a reverse vortex-like flow of the chyme 2
. Bacteria are therefore exposed to a harsh environment which requires sophisticated adhesive tools, i.e. the pili, to maintain stable adhesion.
CFA/I pili that adhere to the intestinal epithelium are approximately 1 μm long helical filaments with a diameter of 7.4 nm and 3.17 pilin subunits per turn of the helix 3
. The major pilin subunit, CfaB, is an approximately cylindrical protein that comprises seven beta strands in an IgG-like structure 4
. The N-terminal strand of one subunit fills a hydrophobic groove of the preceding subunit, thereby producing strong non-covalent interactions along the filament. This helical filament architecture is also seen in P-pili and type 1 pili expressed on uropathogenic E. coli
(UPEC) that infect the bladder and may reach the upper urinary tract and kidneys 5; 6
, as well as type 3 pili expressed by Klebsiella pneumoniae
that cause respiratory tract infections 7
, and E. coli
S pili, which are correlated to neonatal meningitis and urinary tract infections 8
. Adhesion pili assembled via this “donor strand exchange” mechanism 9
provide effective damping against external shear forces by unwinding of their quaternary structure while leaving the tertiary structure intact. This property provides distribution of the shear forces amongst several attached pili and thereby increases the adhesion lifetime 10; 11
. Thus, extension of pili by unwinding of the helical filament allows for motion without breaking the binding structure. Since CFA/I pili are similar in architecture to UPEC-expressed pili 3
we believed that CFA/I pili also were capable of unwinding their quaternary structure and also regaining their original structure after exposure to force, in a similar way to what has previously been found for other pili 8; 12; 13
In this work, we used data from three methodologies in order to characterize and elucidate the function of CFA/I pili under force exposure. Force measuring optical tweezers (FMOT) were used to measure the force required to unwind an individual pilus at a single organelle level. Data were also collected to assess bond kinetics during unwinding. The interactions between adjacent layers responsible for pilus stability were modeled and analyzed in this study using the quaternary structure and orientation of subunits determined previously by a hybrid approach that combined crystallographic data with results from electron microscopy (EM)14
. Finally, the unwinding and retraction biomechanics were modeled by Monte Carlo simulations and fitted to the data.
Our results strongly suggest that the force needed for pilus unwinding is a function of both subunit-subunit interactions and the pitch of the subunits. The limited subunit-subunit interactions as well as their horizontal subunit orientation relative to the normal direction of the applied force lead to the low forces required for CFA/I pili's filament unwinding. Thus, physical properties of the pilus filament facilitate the sustained adhesion of ETEC bacteria in the gut, and thereby facilitate initiation of diarrheal disease.